Modular polyketide synthases (PKSs) are remarkable multifunctional enzyme assemblies that synthesize numerous structurally complex and medicinally important natural products. Over the past decade, the 6- deoxyerythronolide B synthase (DEBS) has established itself as the best-understood modular PKS. In part due to the sustained support of this grant, it is now possible to interrogate the protein chemical, enzymological and biosynthetic properties of DEBS with unrivaled power. Notwithstanding this progress however, we are only just beginning to uncover the structural and mechanistic secrets of DEBS. During the next 5-year period, we will strive to obtain a fundamental and clear understanding of how DEBS controls electrophilic and nucleophilic substrate specificity as well as chain elaboration in each of its six modules. As has been the case in both previous funding cycles, our studies will fully exploit our strong and growing foundation of molecular biological, protein chemical, enzymological, structural biological and biosynthetic tools, and are also expected to yield new macrolide products, Perhaps most significantly, given the status of DEBS as an prototypical modular PKS, it is likely that such insights will be quickly tested in the context of other PKSs and, if validated, used to expand the pool of "unnatural" natural products. The following Specific Aims are proposed for the next 5 years: 1. Developing quantitative enzymological assays to interrogate DEBS modules 1 and 4, the only modules of DEBS for which kinetic assays are not yet available; 2. Dissecting and engineering the specificity of individual DEBS modules toward unnatural nucleophilic substrates (extender units); 3. Analyzing and manipulating the balance between intermodular chain transfer and C-C bond formation in the catalytic cycle of DEB S; 4. Identifying new principles of protein-protein interactions in DEBS, and elucidating their role in maintaining structural integrity and/or facilitating catalysis; 5. Solution NMR, X-ray crystallographic, and electron microscopy studies on DEBS and its components; and 6. Translating insights from Specific Aims 1-4 to produce selected new macrolides.